Alteration of Fc-fusion protein serum half-lives by mutagenesis

Abstract

The present invention provides for a modified Fc-fusion protein in which at least one amino acid from the heavy chain constant region selected from the group consisting of amino acid residues 250, 314, and 428 is substituted with another amino acid which is different from that present in the unmodified Fc-fusion protein, thereby altering the binding affinity for FcRn and/or the serum half-life in comparison to the unmodified Fc-fusion protein.

1. A modified Fc-fusion protein comprising amino acid residues 250 and 428 that differ from the residues present in an unmodified Fc-fusion protein by amino acid residue 250 being glutamic acid or glutamine and amino acid residue 428 being leucine or phenylalanine, and wherein amino acid residues are numbered by the EU numbering system, and the Fc region of the modified Fc-fusion protein is of IgG isotype. 
     
     
       2. The modified Fc-fusion protein according to  claim 1 , wherein the unmodified Fc-fusion protein comprises an Fc region of a human antibody. 
     
     
       3. The modified Fc-fusion protein according to  claim 2 , wherein the human antibody is selected from the group consisting of human IgG1, IgG2, IgG2M3, IgG3 and IgG4 molecule. 
     
     
       4. The modified Fc-fusion protein according to  claim 1  wherein
 (a) amino acid residue 250 is glutamie acid and amino acid residue 428 is phenylalanine; or 
 (b) amino acid residue 250 is glutamine and amino acid residue 428 is phenylalanine; or 
 (c) amino acid residue 250 is glutamine and amino acid residue 428 is leucine. 
 
     
     
       5. The modified Fc-fusion protein according to  claim 1  wherein the modified Fc-fusion protein has a higher affinity for FcRn at pH 6.0 than at pH 8.0. 
     
     
       6. An Fc-fusion protein comprising an Fc region of a naturally occurring class IgG antibody, provided that amino acid residues 250 and 428 differ from the residues present in an unmodified Fc-fusion protein by amino acid residue 250 being glutamic acid or glutamine and amino acid residue 428 being leucine or phenylalanine, and wherein amino acid residues are numbered by the EU numbering system. 
     
     
       7. The Fc-fusion protein according to  claim 6 , wherein said naturally occurring antibody is a human antibody. 
     
     
       8. The Fc-fusion protein according to  claim 6 , wherein said naturally occurring class IgG antibody is selected from the group consisting of a human IgG1, IgG2, IgG3 and IgG4 molecule. 
     
     
       9. The Fc-fusion protein according to  claim 6 , wherein amino acid residue 250 from the Fc region of the antibody is glutamine. 
     
     
       10. The Fc-fusion protein according to  claim 6 , wherein amino acid residue 428 from the Fc region of the antibody is leucine. 
     
     
       11. A modified Fc-fusion protein of  claim 1  with an in vivo mean elimination half-life at least about 1.3-fold longer than that of the unmodified Fc-fusion protein. 
     
     
       12. The modified Fc-fusion protein of  claim 4  with an in vivo mean elimination half-life at least about 1.3-fold longer than that of the unmodified Fc-fusion protein. 
     
     
       13. A method for altering FcRn binding affinity and/or serum half-life of an Fc-fusion protein, said method comprising selecting amino acid residues 250 and 428 present in the Fc region of an Fc-fusion protein, and substituting amino acid residue 250 with glutamic acid or glutamine and amino acid residue 428 with leucine or phenylalanine, and wherein the amino acid residues are numbered by the EU numbering system and the Fc region is of IgG isotype. 
     
     
       14. A method of producing a modified Fc-fusion protein with an altered binding affinity for FcRn and/or an altered serum half-life as compared with the unmodified Fc-fusion protein, said method comprising:
 (a) preparing an expression vector comprising a suitable promoter operably linked to DNA encoding at least an Fc region of an IgG heavy chain comprising amino acid residues 250 and 428 that are substituted with glutamic acid or glutamine at amino acid residue 250 and leucine or phenylalanine at amino acid residue 428, wherein the amino acid residues are numbered by the EU numbering system; 
 (b) transforming host cells with said vector; and 
 (c) culturing said transformed host cells to produce said modified Fc-fusion protein. 
 
     
     
       15. The method according to  claim 14 , wherein:
 (a) amino acid residue 250 is substituted with glutamic acid and amino acid residue 428 is substituted with phenylalanine; or 
 (b) amino acid residue 250 is substituted with glutamine and amino acid residue 428 is substituted with phenylalanine; or 
 (c) amino acid residue 250 is substituted with glutamine and amino acid residue 428 is substituted with leucine.